The comparative perspective of phytochemistry and biological properties of the Apiaceae family plants

Despite the availability of numerous reports on the discovery of medicinal plant compounds and their properties, one may encounter contradictory results released by these reports at the level of plant families and even within species. To establish an accurate perspective of the Apiaceae family, this study examined the fruit essential oil and methanolic extract of wild and common species of this family. According to the measurement of the antioxidant property in the methanolic extract of the fruits using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, Ferula gummosa, Pimpinella anisum and Cuminum cyminum have high power in inhibiting free radicals. However, Bunium persicum had the strongest DPPH radicals inhibitory potential among all essential oils. The results of antimicrobial tests and their classification analysis showed that C. cyminum and B. persicum fruit essential oil with a high amount of cuminaldehyde had the most antibacterial properties. At the same time, the antifungal properties of H. persicum essential oil (rich in aliphatic ester) were stronger than those of the all the studied plants. Also, the essential oils of F. gummosa and Kelussia odoratissima had favourable antimicrobial properties compared to other studied plants. The investigation of the bacterial structure by scanning electron microscope confirmed the effect of the applied essential oils dose and their antibacterial potential. In general, for the first time, this paper determined the biological values of the fruit essential oil of some wild plants, such as K. odoratissima and H. persicum. Besides, in vitro examination and the mathematical models provided a suitable classification, which makes a comprehensive view in terms of the properties of the Apiaceae family.

Methanolic and EOs extraction of fruits. The essential oil of each plant was extracted several times using distillation with water by the Clevenger apparatus. The essential oils of each plant were stored at 4 °C in a dark and closed container in a dark place until use. The methanolic extract was obtained from the fruit (9 g) by a Soxhlet extractor with methanol solvent. Finally, the extract was purified by a rotary evaporator. To make the mother methanolic extract based on the amount of fruits, all the pure extract was again diluted with methanol to 30 mL (300 mg fruit/mL) and stored at 4 °C in dark and sealed containers. At the time of measuring the antioxidant properties, different concentrations (100, 50, 25, 12.5, 6.25 and 3.125 mg fruit/mL) of the main methanolic extract were prepared based on the amount of fruit extracted from the final solution volume.
Total phenolic and flavonoid content. The test solution for estimating the total phenolic content of each fruit contained methanolic extract and Folin-Ciocalteu reagent to which sodium bicarbonate was added after placing in Bain-marie. The test solution for estimating total flavonoid content also contained aluminum chloride, methanolic extract, potassium acetate, and distilled water that was placed in Bain-marie at 25 °C. Finally, the total phenolic content was measured according to the absorbance of the test solution at a wavelength of 760 nm and based on the absorbance of standard concentrations of gallic acid equivalent (GAE) 28 . The total flavonoid content was also measured based on the absorbance of test solutions at 415 nm using a spectrophotometer (Lambda 210 EZ) according to the absorption of standard concentrations of quercetin (quercetin equivalent or QE) 29 . The results are reported based on the mean of 3 replications with ± standard error.
DPPH radical scavenging activity. The antioxidant power of extracts and essential oils was reported based on the concentrations of the extract or essential oil scavenging 50% of DPPH radicals (IC 50 ). To this end, 1 mL of different concentrations of the sample was added to 0.25 mL of 0.2 mM DPPH solution. Accordingly, the absorbance of each sample was read at 517 nm using the spectrophotometer after 30 min of rest at room temperature and in the dark 30 . The results were reported based on means of 3 replications with ± standard error.
Reducing power. To measure the reducing power, 2.5 mL of K 3 Fe(CN) 6 10%, 2.5 mL of 0.2 M potassium phosphate with pH 6.6, and 1 ml of different concentrations of the sample were mixed. In addition, 2.5 mL of Trichloroacetic acid (10%) was added to the resulting mixture after its placement in Bain-marie at 50 °C for 25 min and was then centrifuged. Finally, the test solution containing 2.5 ml of distilled water, 0.5 mL of 0.1 ferric chloride, and 2.5 mL of the centrifuged mixture was read by the spectrophotometer. The final report was based on a concentration of the samples and was equal to 0.5 at a wavelength absorption of 700 nm (EC 50 ) 31 .
Analysis of the essential oil. Gas chromatography-mass spectrometry (GC-MS) analyses were performed with an Agilent 7890 A gas chromatograph equipped with a HP-5MS fused silica column (30 m × 250 μm coated with 5% phenyl methyl silicone, 95% dimethylpolysiloxane, 0.25 μm film thickness), interfaced with an Agilent mass selective detector 5975C inter MSD. Oven temperature was programmed to rise from 60 to 220 °C at a rate of 3 °C/min; and the transfer line temperature was 260 °C. The carrier gas was Helium with a flow rate of 30.6 cm/s and a split ratio of 1:100. Mass scan range from 40 to 300 a.m.u. The identification of oil components was assigned according to the retention index of each GC-MS compound. For measuring the content of each compound, the spectra were considered to be simultaneous if the similarity index was high (%95%). The Kovats index (KI) was also calculated according to compatible Kovats indices (KI) on databases (WILEY, NIST, and PHEROBASE).
Microbial strains. Antimicrobial 32 . Bacterial and fungal suspensions were cultured in Petri dishes containing sterile food media to evaluate the antimicrobial properties based on disk Agar diffusion. It should be noted that Mueller hinton agar (MHA) and Sabouraud dextrose agar (SDA) were used for bacteria and fungi, respectively. The suspensions of microorganisms were prepared freshly and on a daily basis for the tests. The sterile Mueller Hinton Broth (MHB) medium was used for bacteria and Sabouraud dextrose broth (SDB) for fungi. Incubation conditions for bacterial suspension included 37 °C for 24 h. Its concentration was based on 0.5 McFarland Turbidity Standard. In preparing the fungus suspension, it was first prepared in a petri dish containing 7-day SDA Spore Bank. The suspension was then prepared by sterile 0.85% saline solution from the spore bank (~ 10 5 spore. mL −1 ), and 15 µL of EOs were added to the blank disks in each petri dish. For the better diffusion of the biomolecules of the essential oil, each petri dish was first exposed to 4 °C for 4 h. The incubation was then performed on bacterial samples at 37 °C for 24 h, yeast samples at 28 °C for 48 h, and fungal samples at 28 °C for 72 h. The antimicrobial activity of each essential oil was determined by a caliper based on inhibition zone (mm) measurements. The results were reported based on means of 3 replications with ± standard error. The controls of ciprofloxacin (5 μg per disc) and genatamicin (10 μg per disc) of Padtanteb, Iran were used for bacteria; and the Voriconazole (1 μg per disc) of RIDACOM brand was used for fungi and yeast. Human and animal participants. This article does not contain any studies with human or animal subjects.

Results and discussion
Secondary metabolite and antioxidant activity of fruits. The amount of total phenol and flavonoids in the extracts of the studied species showed a significant difference ( Table 1). The antioxidant properties of methanolic extracts were determined using DPPH and reducing power methods. In many studies, the amounts of antioxidant properties were reported based on the pure solid extract obtained by various solvents that were then purified. In the present study, the measurement of antioxidant properties was based on milligrams (mg) of the extracted fruit since the plants were from different species. Thus, different weights of the solid extract were obtained in the extraction of each species based on a certain amount of the fruit. Therefore, determining the level of antioxidant activity solely based on the weight of the pure extract (solid extract) may not be a reasonable basis for determining the level of the antioxidant activity of the fruit in a comparative study. This is so because a particular pure plant extract may have considerable antioxidant levels. However, the weight ratio of the pure extract per mg of fruit and its antioxidant activity based on fruit weight can be lower than those of other plants. Also, since fruits of all species are used in powder as spices and traditional medicines, the comparison of antioxidant activity in fruits compared to pure extract can be more functional in the present study.  (Table 1). Previous studies have demonstrated that levels of phenolic and flavonoid compounds in a plant species are directly and strongly associated with antioxidant powers such as reducing power and DPPH radical scavenging 34,35 . Therefore, the higher levels of TPC and TFC in F. gummosa and P. anisum fruit compared to the other plants were the main reasons for its antioxidant power. On the other hand, C. cyminum contains a smaller amount of TPC and TFC than some studied species; however, the fruit of this plant had the highest antioxidant activity against DPPH radicals with IC 50 of 9.48 mg/mL (Table 1). These results can be attributed to the differences in the types of flavonoid and phenolic compounds of the different plants (Table 1). Different phenolic and flavonoid compounds have different antioxidant powers 36,37 .
Chemical composition and density of essential oils. According to Tables 2 and 3, some differences were revealed in the chemical profiles of essential oils extracted from all the fruits of the plants. The highest range of compounds was identified in K. odoratissima (31 compounds), and the smallest one was related to P. anisum (8 compounds). On the other hand, Table 4 shows that the density of each essential oil is different due to its unique chemical profile. The chemical type of F. gummosa, P. crispum and A. graveolens essential oils were mostly monoterpene hydrocarbons and oxygenated monoterpenes (Table 2). Also, the density ratios of these plants were about 0.859, 0.826 and 0.873 mg/µL, respectively ( Table 4). The main compounds of A. graveolens essential oil included limonene (monoterpene hydrocarbons), carvone (oxygenated monoterpenoids) and dill apiole (phenylpropanoids) in amounts of 33.12, 54.2 and 4.04%, respectively. α-pinene and β-pinene were the main terpene components identified in the essential oils of the two plants, and they constituted 22.83% and 48.36% of the total essential oil in F. gummosa, and 45.3% and 29.70% of the total essential oil in P. crispum, respectively ( Table 2). δ-3-carene was also a member of the monoterpene hydrocarbon family and accounted for 10.69% of F. gummosa essential oil ( Table 2). Phenylpropanoids constituted 11.22% of the essential oil of P. crispum. Indeed, myristicin was the most important phenylpropanoid in P. crispum (9.34%) ( Table 2). In addition to high amounts of monoterpene hydrocarbons and oxygenated monoterpenoids, C. cyminum and B. persicum also had considerable amounts of aldehyde. The highest aldehyde content in these essential oils belonged to cuminaldehyde and γ-terpinen-7-al, which were about 13.92% and 15.34% in C. cyminum and 41.84% and 3.32% in B. persicum. Also, their main original monoterpene hydrocarbons were p-cymene, β-pinene, and γ-terpinene (Table 2). In comparison to C. cyminum (0.914 mg/µL), the density of B. persicum (0.876 mg/µL) essential oil was smaller ( Table 2). sesquiterpene hydrocarbon is another fundamental family of the chemical profile of essential oils. They comprise about 67.2% K. odoratissima essential oil. The major chemical compounds of K. odoratissima essential oil encompassed δ-cadinene, selinene, germacrene D, and γ-elemene. limonene (monoterpene hydrocarbon), and Z-ligustilide (phthalides) were the two other major components constituting 8.74% and 15.18% of the essential oil of this plant, respectively (Table 3). trans-anethole is a phenylpropanoid whose amounts in the fruits of P. anisum and F. vulgare were 75.43 and 63.35% of essential oil, respectively (Table 3). Also, the essential oil of these plants had the highest mass-to-volume ratio (mg/μL) compared to other plants (Table 4). Other important compounds of these plants comprised limonene (10.13%), carvone (9.15%) in P. anisum, and fenchone (16.75%) in F. vulgare essential oils ( Table 3). The classes of the compositions of H. persicum essential oil were totally different. In addition, this essential oil has the lowest density  Antioxidant activity of fruits essential oil. We presented essential oil antioxidant activity through reducing power and DPPH methods. Through DPPH methods (IC 50 ), the antioxidant potential of F. gummosa and P. crispum essential oils was evaluated at about 40.7 and 22.13 mg/mL (Table 4). Also, the reducing powers (EC 50 ) of F. gummosa and P. crispum essential oils were estimated at about 83.38 and 183.33 mg/mL. Indeed, F. gummosa had a weaker DPPH radical scavenging capacity and stronger reducing power than P. crispum, while both of them contained high amounts of β-pinene and α-pinene (Table 4). Usually, the biological activity of essential oils varies in different plant species. At the same time, the difference in enantiomers content of a compound with different biological activity, such as α-Pinene and β-pinene enantiomers, have distinct properties 46 .
The lowest antioxidant activity of essential oils belonged to F. vulgare. Also, H. persicum essential oil had a weaker antioxidant power than the most studied plants. The lowest consumption of essential oils for inhibiting 50% of DPPH (IC 50 ) is related to B. persicum (Table 4). Therefore, in terms of scavenging DPPH radicals, the B. persicum essential oil (IC 50 : 10.81 mg/mL) has the highest potential. Afterwards, C. cyminum and P. anisum had a stronger antioxidant activity than others based on the DPPH method. The ranges of IC 50 , such as 114.87 to 287.56 µg/mL 45 and 0.384 to 0.809 mg/mL 47 were presented for the DPPH free radical scavenging capacity of the P. anisum essential oil. These findings are inconsistent with that of the present study. In a report released by Das et al. 48 , the IC 50 of P. anisum (Varanasi, India) essential oil was equal to 12.31 µL/mL (for DPPH free radical scavenging), which is consistent with the finding obtained in this study (IC 50 = 19.56 mg/mL), due to the range of density of the P. anisum essential oil (0.932 g/mL or mg/µL). The slight difference between them was due to differences in the content of constituents of essential oils. In previous studies, IC 50 was reported to range from 7.9 to 13.6 mg/mL for DPPH scavenging capacity of C. cyminum essential oil 49 and is in the acceptable range of the current study. It has been reported that among cumin essential oil compounds, the DPPH radical inhibitory power of γ-terpinene (IC 50 = 22.73 mg/mL) was higher than those of other compounds such as cuminaldehyde (IC 50 = 105.36 mg/mL), β-pinene (IC50 = 135.55 mg/mL) and p-cymene (IC 50 = 122.25 mg/mL), and the scavenging power of the C. cyminum essential oil based on IC 50 was 35.94 mg/mL 50 . Therefore, the antioxidant properties of essential oils such as C. cyminum and B. persicum can differ from the main pure compounds due to the interaction between the components and the chemical structure of the essential oil profile. Based on the DPPH method, the antioxidant power of K. odoratissima essential oil was weaker than those of all species (except F. vulgare). However, this plant has the highest reducing power with EC 50 of 43.22 mg/Ml, while both of these methods are based on measuring the electron-donating capacity of an antioxidant. Therefore, there should not be huge contradictions in determining the antioxidant potential of several chemical compounds (like essential oil) in these methods. In some cases, the results of the reducing power method have little in consonance with the DPPH method regarding the antioxidant properties of these plants essential oils. For instance, according to the DPPH results, the essential oils of C. cyminum, B. persicum, and P. anisum show a high antioxidant power. However, the results obtained from their reducing power indicate a lower antioxidant power than some plants like F. gummosa and K. odoratissima (Table 4). In the reducing power methods, all reaction solutions were watersoluble. Owing to the nature of essential oils as nonpolar and hydrophobic molecules, the essential oil concentration was prepared in a non-aqueous solvent like methanol. When the reaction solution and different essential oil concentrations were mixed, an emulsion was formed to evaluate the reducing power of essential oils. It has been proven that different types of chemical compositions of essential oils affect the quality and stability of emulsions formed 51,52 . Therefore, unlike the DPPH method, the reducing power of the essential oil, in addition to its antioxidant power, is influenced by the quality and stability of the emulsion in different plants. This approach makes some misinterpretations in the antioxidant power of essential oils in this method. For this reason, unlike the antioxidant report of the methanol extract of the fruit (Table 1), the antioxidant power results of the essential oil via DPPH and power reduction methods could not be matched. Furthermore, the evaluation of the reducing power of essential oils at concentrations above 200 mg/mL was inestimable due to the instability of the emulsions created in the reaction solution. On the other hand, in the essential oils of A. graveolens, P. anisum, F. vulgare, and H. persicum, the increasing trend of reducing power was insignificant with an increasing concentration; therefore, EC 50 (absorption 0.5) was not calculated (Table 4). In the previous study, it has been reported that in   Antimicrobial activity. In the current study, positive controls were Gentamicin and Ciprofloxacin for antibacterial properties and Voriconazole for antifungal properties. As shown in Table 5, P. aeruginosa is the most resistant bacterium to the used chemical antibiotics. Furthermore, P. aeruginosa has more resistance to essential oils than other bacteria. P. aeruginosa species has a high resistance to external factors due to having a lipopolysaccharide and phospholipid membrane 54 . Also, the lipopolysaccharide membrane plays an essential role in the resistance of this bacterium to hydrophobic antibacterial compounds (e.g., essential oils) 55 .
In general, there are no detailed reports on the antimicrobial properties of some wild plants, such as F. gummosa, H. persicum and K. odoratissima. However, in Table 6, a brief report of the range of antimicrobial properties of the essential oils of Apiaceae family plants was obtained based on MIC and MBC. What is evident in the presented table is that the range of estimated values is vastly contradictory. In fact, based on a general summary of previous research results, it is impossible to distinguish the antimicrobial power of each plant's essential oil, either separately or comparatively within the family. In previous studies, the range of MIC and MBC values exists as values with units of µg/mL and mg/mL in some species (Table 6). Although the results can be different due    www.nature.com/scientificreports/ to the difference in the bacterial strains and the chemical profile of the plants, many of the observed differences are beyond these factors. Due to the liquid nature of essential oils, it is common to report MIC and MBC values as µL/mL. However, special attention should be paid to the density of essential oils to express MIC and MBC based on µg/mL and mg/mL. As presented in Table 4, each essential oil has a different density according to the its constituent compounds. For example, according to Table 7, the MIC value in the essential oil of H. Persicum and F. vulgare plants against A. flavus was estimated as 1 mg/ML. According to the density of the essential oil of H. Persicum (0.766 mg/µL) and F. vulgare (0.990 mg/µL), this value is equivalent to 1.30 and 1.01 µL/mL, respectively. The lack of attention to the density and the approach expressed regarding unit conversion are the main reasons for the difference in various reports. In addition, a numerical range regarding the antimicrobial properties of the main purified compounds related to the essential oils of this family, such as α-pinene, limonene, carvone, trans-anethole and ligustilide has also been presented in Table 6. This can provide an overview of the range of antimicrobial effects of the main essential oil compounds of this family in the form of MIC and MBC values. In this study, according to the results of a wide range of effective concentrations in previous studies, the range of concentrations from 0.005 mg/mL (equivalent to 5 µg/mL) to 96 mg/mL was tested to enable the conduct of accurate evaluation and measurements. According to Tables 7 and 8 26 reported that the range of MIC of A. graveolens essential oil (containing 44.61% limonene, 28.02% carvone and 19.98% dillapiole) was higher than 10 mg/Ml. While the reported chemical profile was partly different from that of the current study, especially in terms of dillapiole content. limonene constitutes 33.12% of the essential oil of A. graveolens and 8.74% of the essential oil of K. odoratissima. However, the antibacterial properties of this compound through the MIC method were reported to be approximately 16 µL/mL against E. coli 72 . In another study, the reported MIC values of 26.25 and 52.5 mg/mL for E. coli and S. aureus were claimed, respectively 73 . Carvone was another main A. graveolens essential oil compound (54.3%) and its MIC against S. aureus (ATCC 6538) and B. subtilis (ATCC 6633) with the same strains of the current study were evaluated to be about 5 and 10 mg/mL 22 . The current result also demonstrates that the inhibitory and bactericidal power of A. graveolens essential oil against S. aureus is better than B. subtilis; and the range of MIC and MBC has consonance with the biological activity of its main compounds. In the case of limonene, in addition to the antibacterial effects of the compound itself, high synergistic effects were observed between the compound and chemical antibiotic compounds against several bacterial species such as E. coli and S. aureus 74 . The presence of limonene, along with other main components of K. odoratissima essential oil, may increase the synergetic effects and consequently increase the antibacterial properties. As it was mentioned above, there are no specific reports about the antibacterial properties of K. odoratissima and H. persicum; however, the biological properties of any essential oil are directly related to its components. For instance, K. odoratissima contains a high amount of Z-ligustilide and its MICs were evaluated to be 1, 1, 8 and 10 mg/mL against B. subtilis, S. aureus, E. coli and P. aeruginosa, respectively (Table 8). Previously, it has been reported that Z-ligustilide compound purified from Cnidium officinale essential oil had high antibacterial properties, and its MIC against S. aureus was 1 mg/mL 70 . The amount of MIC in H. persicum essential oil was estimated to be 8, 10, 8, and 24 mg/mL against B. subtilis, S. aureus, E. coli and P. aeruginosa, respectively (Table 8). Its essential oil consists of high amount of acetate composition and octanol. These compounds such as octyl acetate and octanol were factors of antibacterial properties in the Zosima absinthifolia plant 75    www.nature.com/scientificreports/ oil up to 96 mg/mL had no antibacterial power on the tested bacteria (Tables 8). Furthermore, these essential oils inhibited the lowest zone (Table 7). Even the essential oil of P. crispum and P. anisum against B. subtilis bacterium did not form any inhibition zone. Approximately 75 and 63% of the essential oil of P. anisum and F. vulgare were trans-anethole. In the past, it has been determined that trans-anethole isolated from the Croton zehntneri plant did not have antibacterial properties against S. aureus, P. aeruginosa, and E. coli bacteria 76 . Also, the previous evaluation of trans-anethole activity against S. aureus indicated that this compound had a weak antimicrobial potential 68 . Presumably, trans-anethole has a non-toxic effect on bacteria so it can be the reason for the lack of antibacterial responses in the P. anisum and F. vulgare essential oil. Similar to the results of the current study, Fitsiou et al. 77 studied the essential oils of several medicinal plants and found that the P. anisum essential oil did not have any antibacterial activity. On the contrary, there were reports of antibacterial properties of the essential oil of this plant on the same bacteria, MIC in the range of 0.06 to 0.25 µg/mL 78 and 2.0 to 3.0 µg/mL 60 were reported, which were inconsistent with the results of this study. According to the results of Table 2, the predominant compounds of F. gummosa and P. crispum essential oils are two common compounds of α-pinene and β-pinene. However, there are significant differences between www.nature.com/scientificreports/ antibacterial properties of these essential oils in terms of MIC, MBC and inhibition zone traits. The weak antibacterial properties of P. crispum essential oil have previously been revealed 79 . In contrast, the amount of inhibition zone of F. gummosa essential oils indicated strong antibacterial properties. Similarly, these estimations indicate its MIC and MBC value against B. subtilis, S. aureus, and E. coli was 2 mg/mL. The possibility of differences in the enantiomeric profile of the main components of the essential oils of these plants is a major reason for this difference. It was reported that differences in levels of enantiomeric compositions of α-pinene ((1R)-(+)-α-pinene and (1S)-(−)-α-Pinene) in Juniperus communis essential oil could increase its lethality effects by 4 to 16 times. It has also been reported that (−)-α-pinene, as an enantiomeric composition of α-Pinene, has had no antibacterial or antifungal properties; however, (+)-α-pinene has had strong antimicrobial properties 80 . Therefore, the amounts of pinene detected in the essential oils of the two different plants, including F. gummosa and P. crispum, might have different chemical reactions due to the differences in the enantiomeric compositions of Pinene. Also, the most enantiomeric composition of α-pinene in F. gummosa essential oil has been reported to be (1R)-(+)-α-pinene 38 .
Interactions and combined effects of all the essential oil components could be another reason for the differences in the properties of F. gummosa and P. crispum essential oils. This is due to the fact that the final properties of each essential oil are affected by all essential oil compounds and their antagonist and synergetic effects. For example, it is reported that (−)-α-pinene alone does not have high antibacterial properties; however, its use in combination with chemical antibiotics increases their antibacterial power by several hundred times 65 . Therefore, the interaction of the chemical components of the essential oil leads to differences in their properties due to the different chemical profile of each species. The δ-3-Carene compound is an active compound against bacteria with a MIC of about 0.16 g/L (equivalent to 0.16 mg/mL) against E. coli, B. cereus, and S. aureus 69 . It has also been determined that the reason for the high antibacterial power of 3-Carene compound can be attributed to the multiple mechanisms of its destructive effects on the morphological, physiological and molecular levels of bacteria. At the morphological level, this compound causes changes in the natural shape and structure of bacteria, disruption and destruction of bacterial membrane integrity, and leakage of intracellular substances. At the     www.nature.com/scientificreports/ physiological levels, it reduces the metabolic function by impairing the ATP breakdown, intracellular Tricarboxylic acid cycle, and Glycolysis-related enzymes. At the molecular level, this compound binds to DNA, disrupts gene expression, and as a result, increases the antibacterial effect 81 . Therefore, the presence of this compound in F. gummosa essential oil, along with other essential oil components, can be a significant reason for the higher potency of its antibacterial properties than P. crispum essential oil (Tables 7, 8). The Antibacterial properties of C. cyminum and B. persicum were higher than others in all bacterial species based on the inhibition zone, MIC, and MBC. The concentration ranges of MIC, MBC, and inhibition zone for C. cyminum essential oil were 0.5-4, 1-10 mg/mL, and 24.88-37.23 mm and for B. persicum were 0.5-4, 1-6 mg/mL, and 25.23-39.33 mm, respectively (Tables 7, 8). In relation to the antibacterial properties of C. cyminum and B. persicum essential oils in the current study, the range of MIC and MBC had consonance with previous research findings. However, the expected difference could be attributed to differences in essential oils compounds and differences in the resistance of bacterial strains. Among the studied plants, H. persicum, C. cyminum and B. persicum essential oils had high antifungal activity against both A. flavus and C. albicans. In contrast, the essential oils of A. graveolens, P. anisum and F. vulgare had the lowest antifungal activity against C. albicans (Tables 7, 8). The essential oils of F. gummosa and P. crispum showed inhibition against A. flavus in 4 mg/mL concentration, but it seems that they had the lowest fungicidal properties against this fungus. Generally, it has been founded that since plant essential oils have hydrophobic components, they degrade and disrupt the membrane fluidity, change ergosterol biosynthesis, increase leakage, and change pH in the cytosolic space. Thus, they cause damage to cell organs and lead to the death of fungal and yeast cells 5 . In addition, the results showed that different compounds of the essential oils of each plant have different effects on different microorganisms. For example, it has been reported that α-pinene has no strong fungicidal effect against A. flavus 82 . According to the results of one study on the fungicidal effect of α-Pinene, the estimated amount of the MFC for A. flavus for this compound was approximately 15 times higher than the MFC for C. albicans 80 . Considering the amount of pinene in the essential oils of P. crispum and F. gummosa, it can be a reason for the superiority of these essential oils in the inhibition and lethality power of C. albicans compared to A. flavus. According to another report, cuminaldehyde has a high potency in damaging fungal hyphae, reducing ergosterol biosynthesis, growth inhibition, and aflatoxin production in A. flavus, as the MIC of fungal growth in this compound is about 0.64 μL/mL (equal to 0.62 mg/mL considering a density of about 0.97 g/mL for this compound) 83 . Previously, Das et al. 48 have indicated the power of P. anisum essential oil in inhibiting A. flavus. anethole has been the main component of a P. anisum and F. vulgare essential oil. It has also been previously reported that the antifungal properties of Illicium verum and F. vulgare essential oils can be attributed to the high amount of anethole 84,85 . Related research findings have revealed that the membranes and walls of bacteria are destroyed by plant essential oils. However, other destructive effects such as cytoplasmic coagulation, cytoplasmic content leakage, and protein damage have also been reported against bacteria with essential oil application 86 . One of the reasons for the destruction of the bacterial membrane is the hydrophobic nature of essential oils, which disrupts the two phospholipid layers of the bacterial membrane, although the destructive effects of each essential oil on bacteria www.nature.com/scientificreports/ may be different depending on the type and composition of each essential oil 6 . All the SEM images show the effect of membrane degradation on both bacterial species with the application of H. persicum and C. cyminum essential oils (Figs. 1, 2). In SEM images, some membrane destruction can be detected in both species of bacteria with the use of H. persicum essential oil, but the SEM imaging of C. cyminum show that the power of C. cyminum essential oil in membrane destruction is much more intense. In addition, the effects of C. cyminum on the destruction of each of the two bacteria, especially B. subtili, can be regarded as a mass of damaged cell membranes. Some bacterial cells with disrupted membranes can be observed in A. graveolens treatments. But the effects of A. graveolens essential oil were lower than those of H. persicum and C. cyminum. In addition, no membrane degradation by P. crispum essential oil was not observed. Nevertheless, the essential oil of this plant caused morphological changes in bacteria, including changes in the size of the bacteria and shrinkage of the bacterial membrane, especially E. coli. (Figs. 1, 2).
Principal component analysis (PCA) and agglomerative hierarchical clustering (AHC) on essential oils' profiles and properties. AHC analysis is a statistical approach to categorizing the tested factors based on their characteristics. In the current research, this analysis was done three times based on essential oil compounds data (Fig. 3a), antimicrobial properties data (Fig. 3b), and data related to essential oil compounds and antimicrobial properties together (Fig. 3c). As presented in Fig. 3a, the essential oils of KO and HP plants are more distinct from other plants in terms of chemical profile, and both of them form a cluster independently. FG and PC with a high level of pinene and BP and CC with a high level of Aldehyde compounds form the other two clusters. Finally, PA and FV with high similarity to each other and together with AG formed a cluster (Fig. 3a). In Fig. 3b, it has been shown that there are three classifications based on antimicrobial properties. The cluster has a group with high antimicrobial properties, including CC, BP, FG and KO; a group with moderate antimicrobial properties, including HP and AG; and a group with weak antimicrobial properties, including FV, PA and PC. Clearly, the best and most accurate classification was obtained based on the analysis of all data related to essential oil compounds and biological activities (Fig. 3c). As a result, the differences between the plants have been determined more precisely in Fig. 3c based on essential oil compounds and biological activities and, thereby, a more complete and accurate view has been presented. The identification of compounds or common tests to evaluate the biological properties of essential oils is a costly, time-consuming, and sometimes dangerous process. On this basis, there is the possibility that a mathematical prediction model in the future can predict the properties of essential oils based on their constituent compounds. Since there are valuable differences and similarities in the grouping of Fig. 3a, b with Fig. 3c. for example, in all three modes of analysis, CC and BP have been placed in one group, and PA and FV have been placed in another group (Fig. 3a-c). This shows that it is possible to determine the potential of antimicrobial properties using modeling in advance, particularly in intra-familial classes based on the chemical composition of these plants. Reyes-Jurado et al. 87 clearly state that common laboratory tests regarding the antimicrobial properties of essential oils are influenced by many variables such as human errors, source of essential oil, extraction method, amount www.nature.com/scientificreports/ of chemical compounds, the volume of inoculation, growth phase of microorganism, the strain of microorganisms to be tested, the culture medium used, the incubation time, the temperature, and the pH of water. Finally, according to the impact of the mentioned cases, there are distinctions and differences in the reports, even about a plant species ( Table 6). As a result, as in the present study, one of the most effective methods to reduce these differences is to examine several plant species simultaneously and use techniques such as SEM to confirm the results of tests that rely solely on visual observations (such as MIC, MBC and inhibition zone). In this regard, the factors causing differences during the tests become uniform and, thereby, accurate comparisons are made with less error. However, the use of a mathematical model seems to be a more appropriate solution. Today, the mathematical modeling of chemical antibiotics is done in very advanced fields to check the effective dose even in the human body 88 . At the same time, there is no report regarding the application of these methods to investigate plant compounds' properties, even in vitro. In the present study, advanced modeling was not applicable due to the small number of tested samples. Therefore, the extent and number of laboratory samples in the future should be considered. Also, the placement of KO and HP in separate groupings in different states in AHC analysis shows that plant species should be selected based on similarities in chemical profiles to design the prediction model in the future. From each chemical profile with similar compositions, there should be several plant species for the conduct of a more accurate modeling. Accordingly, very contradictory chemical profiles should be removed from the prediction model. Another apparent contradiction is the position of FG in these groupings. Indeed, in Fig. 3a, the high level of pinene placed this plant in the same group as PC plant. While in their grouping based on antibacterial properties, the position of these two plants differed (Fig. 3a). In Fig. 3c, the FG plant was also placed in a group alone. It was pointed out that the enantiomers of a compound such as α-pinene have different biological properties. This trend shows that the difference in biological properties of enantiomeric composition can cause errors in the model. www.nature.com/scientificreports/ Therefore, for the accomplishment of more accurate modeling, the enantiomeric composition levels of the main components of the essential oil should be well estimated. Based on the PCA analysis (Fig. 3d) and the location of independent variables (active observation), a high level of similarity between the essential oils of CC and BC plants and FG and PA plants was also observed. In the PCA diagram, the more acute the angle between the two factors, the stronger and more positive the correlation between the two factors. In this regard, the aldehyde compounds in the Apiaceae family can be considered potent bactericidal agents. Additionally, according to the distance of the traits indicating antibacterial properties (active variables) in different plants (active observation), the essential oils of two plants CC and BP (containing a high level of aldehyde compounds), had high antibacterial properties. Furthermore, the ester and alcohol compounds of plant essential oils are highly effective in killing fungi. HP plant essential oil shows the most fungicidal effect on these microorganisms.

Conclusion
Overall, the results of measuring the methanol extract of fruits by DPPH and reducing power methods showed that the fruits of F. gummosa, C. cyminum and P. anisum plants had higher antioxidant properties than other studied plants. According to the DPPH method, the antioxidant properties of essential oil C. cyminum and B. persicum were stronger than others. Considering previous studies and a comparative approach to investigate the properties of 9 plants of the Apiaceae family, the present study determined the potential of these essential oils in terms of antimicrobial properties and effective doses. In this regard, the essential oils of 3 plants, K. odoratissima, F. gummosa, and H. persicum, with their specific chemical profiles, had a favorable potential in inhibiting the growth of various bacteria. The essential oil of H. persicum was more effective than other plants in inhibiting C. albicans and A. flavus. Thus, the essential oil of this plant can be a suitable option for medical applications to inhibit this human pathogen C. albicans and to inhibit fungal food contamination and storage loss of A. flavus. The essential oils of C. cyminum and B. persicum plants had very high antibacterial properties compared to others, hence, both plants are more emphatically recommended for industrial and medical applications. Also, the results of AHC and PCA analysis showed that the creation of mathematical models for predicting the biological properties of essential oils based on their constituents can be effective and it is suggested to pay more attention to this finding in the future.

Data availability
Raw data and derived data supporting the findings of this study are available from the corresponding author on request.